Diva decloaker antigen retrieval solution

For Ki-67 staining, formalin-fixed paraffin-embedded tissue sections were de-waxed and rehydrated, incubated in Diva Decloaker antigen retrieval solution (Biocare) and boiled for 20 min in a pressure cooker. Peroxidase activity was blocked using the Enzyme Block for 15 min (DAKO). Slides were stained with 1:400 anti-Ki-67 (D3B5; Cell signaling) for 1 h followed by another hour of incubation with 1:500 of goat anti-rabbit (Jackson laboratories). Finally, slides were incubated with DAKO DAB substrate (DAKO) for 10 min and counter-stained with hematoxilin, then mounted. For Alcian Blue staining (1% solution, Sigma), slides were stained at room temperature for 30 min and then washed. Cell nuclei were stained with Kernchtrot Nuclear Fast Red stain (1 min). Slides were washed with distilled water, dehydrated with anhydrous alcohol and mounted.

Paraffin blocks were sectioned using a microtome to obtain 4 μm thick sections for immunostaining. The paraffin sections were dewaxed in xylene and hydrated in decreasing concentrations of ethanol. Sections were then incubate in 1 × DIVA Decloaker antigen retrieval solution (Biocare Medical) at 110°C for 15 min using the decloaking chamber (Biocare Medical). Following antigen retrieval, sections were incubated in peroxidazed 1 solution (Biocare Medical) at room temperature for 5 min to quench endogenous peroxidase activity. After blocked with background sniper at room temperature for 10 min, sections were incubated with a monoclonal rabbit anti-human CD3 antibody (0.3 μg/ml; Biocare Medical) in Dako REAL antibody diluent (Dako) at room temperature for 1 h. Sections were subsequently incubated with HRP-labeled goat anti-rabbit IgG polymer (Dako) at room temperature for 30 min. Finally, sections were exposed to liquid DAB+ substrate chromogen system (Dako) at room temperature for 5 min and counterstaining was performed using Gill's hematoxylin (Sigma).